Cell, Vol. 68, 491-505,

February

7, 1992, Copyright

0 1992 by Cell Press

Twin of I-POU: A Two Amino Acid Difference in the I-POU Homeodomain Distinguishes an Activator from an Inhibitor of Transcription Maurice N. Treacy,“t Lorna I. Neilson,’ Eric E. Turner,** Xi He,‘5 and Michael G. Rosenfeld*t * Eukaryotic Regulatory Biology Program fHoward Hughes Medical Institute *Department of Psychiatry SDepartment of Biology University of California, San Diego School of Medicine La Jolla, California 92093-0648

Summary I-POU, a POU domain nuclear protein that lacks two conserved basic amino acids of the POU homeodomain is coexpressed in the developing Drosophila nervous system with a second POU domain transcription factor, W-a. I-POU does not bind to DNA but forms a POU domain-mediated, high affinity heterodimer with M-a, inhibiting its ability to bind and activate thedopa decarboxylase gene. The I-POUICfl-a dimerization interface encompasses only the N-terminal basic region and helices 1 and 2 of the POU homeodomains with precise amino acid and a-helical requirements. twin of I-POU, an alternatively spliced transcript of the I-POU gene, encodes a protein containing the two basic amino acid residues absent in I-POU. Twin of I-POU is incapable of dimerizing with W-a, but can act as a positive transcription factor on targets distinct from those regulated by (X1-a. These findings suggest that the I-POU genomic locus simultaneously generates both a specific activator and inhibitor of gene transcription, capable of modulating two distinct regulatory programs during neural development. Introduction To generate specific cell phenotypes, multiple families of transcription factors act combinatorially in activating and repressing gene programs in organisms as divergent as yeast and mammals (reviewed in Herskowitz, 1989; Mitchell andTjian, 1989; Johnson and McKnight, 1989; Orkin, 1990; Rosenfeld, 1991; Struhl, 1991; Weintraub et al., 1991). Accordingly, heterodimeric interactions between members of a family of transcription factors can serve critical inhibitory, as well asactivating, functions in regulatingprogramsof genetranscription(e.g., Murreetal., 1989; Glass et al., 1989; Baeuerle and Baltimore, 1988; Benezra et al., 1990; Treacyet al., 1991). The membersof the POU domain family of developmentally active transcriptional regulators(Ingrahametal., 1988;Clercetal., 1988; Finney et al., 1988; Ko et al., 1988; Mtiller et al., 1988; Bodner et al., 1988; Scheidereit et al., 1988; Sturm et al., 1988; Burglin et al., 1989; He et al., 1989; Johnson and Hirsh, 1990; Monuki et al., 1990; Okamoto et al., 1990; Scholer et al.,

1990; Suzuki et al., 1990) are characterized by a unique, bipartite DNA-binding domain (reviewed in Herr et al., 1988; Ruvkun and Finney, 1991; Rosenfeld, 1991). The POU domain consists of a 76 amino acid POU-specific (PO&) domain that exhibits the highest degree of conservation, separated by a spacer of variable length from a 60 amino acid variant POU homeodomain (POUHD) exhibiting homology to the classic homeodomain motifs present in many developmental regulators in Drosophilaand all other metazoans (reviewed in Gehring, 1987; Akam, 1987; Scott et al., 1989). Several members of the large POU domain gene family exert critical functions in the appearance of specific cell phenotypes. Pit-l is required for appearance and proliferation of 3 of the 5 distinct cell types of the anterior pituitary gland (Li et al., 1990); uric-86controls the development of several neuronal lineages in Caenorhabditis elegans (Finney et al., 1988; Finney and Ruvkun, 1990); and Ott-3/4 appears to be required for the initial zygotic cell division (Rosner et al., 1991). The POUs domain appears to be required for high affinity, site-specific DNA binding (Sturm et al., 1988; lngraham et al., 1990; Verrijzer et al., 1990) as well as DNA-dependent proteinprotein interactions (Ingraham et al., 1990), while the PO& is critical for DNA binding. The POUHo can also be involved in protein-protein interactions, as the viral aTIF/ VP16 protein interacts with the Ott-1 homeodomain (McKnight et al., 1987; Gerster and Roeder, 1988; O’Hare and Goding, 1988; Stern et al., 1989; Kristie et al., 1989; Kristie and Sharp, 1990). Members of the POU domain gene family have been suggested to be largely monomeric in solution, but Pit-l, Ott-2, and Cfl-a exhibit cooperative DNA-dependent protein-protein interactions on certain cis-active DNA elements (LeBowitz et al., 1989; lngraham et al., 1990; Treaty et al., 1991; Voss et al., 1991). Investigation of POU domain proteins in the developing Drosophila nervous system has revealed a high affinity protein-protein interaction in solution between two POU domain proteins with an important regulatory consequence (Treaty et al., 1991). I-POU, highly homologous over the entire POU domain to the other members of the POU IV class of proteins (Brn-3 and uric-86) lacks two basic amino acid residues in the highly conserved basic cluster at the N-terminal portion of the PO&o, rendering it incapable of binding DNA. I-POU exhibits an overlapping pattern of expression with a second DNA-binding POU domain regulatory protein, Cfl-a, which binds to specific elements in the dopa decarboxylase (Ddc) gene, activating its transcription (Johnson and Hirsh, 1990; Treaty et al., 1991). I-POU forms heterodimers with Cfl-a in solution (Treaty et al., 1991) inhibiting Cf 1 -a from binding specific cis-active elements and transactivating the Ddc gene (Treaty et al., 1991). The affinity of Cfl -a for I-POU exceeds its affinity for the cognate DNAbinding site in the Ddc promoter. Thus, actions of I-POU in the POU domain gene family might be considered to be similar to those of Id, a member of the helix-loop-helix gene

Cell 492

family that serves to inhibit DNA binding of multiple other helix-loop-helix proteins as a consequence of heterodimer formation (Benezra et al., 1990). In this manuscript, we report that the interactions of I-POU and Cfl -a are highly specific, with I-POU failing to interact with any other known member of the POU domain gene family. The regions critical for this high affinity interaction are limited to the basic amino acid cluster at the N-terminusand the first two helices of the PO&. We have identified a novel, alternatively spliced transcript of the I-POU gene that encodes a variant of I-POU, referred to as twin of I-POU (fl-POU), in which the two basic amino acid residues absent within the N-terminus of the POUHD are restored. tl-POU is no longer capable of interacting with Cfl-a, nor does it interact with I-POU. However, tl-POU is capable of specific DNA binding and transactivates reporter genes containing cognate DNA-binding elements that are distinct from the Cfl-a recognition sequence. The I-POU genomic locus therefore simultaneously generates both a specific activator and inhibitor of distinct programs of neuronal gene transcription, differing only by the presence or absence of two basic amino acid residues in the PO&D. Results The POUHD Alone Is Required for Protein-Protein Interaction The dimerization domain responsible A. I-POU

for the I-POUICfl-a

interaction was investigated by generating deletion mutants of both I-POU and Cfl-a (Figure 1). Neither Cfl-a or I-POU N-terminal or C-terminal regions outside the POU domain contained information required for dimerization (Figure 1A, lanes 3 and 4; Figure 1 B, lanes 3, 4, 11, and 12). However, the POU domains of either I-POU or Cfl-a could dimerize readily with either wild-type or the isolated POU domain of the other protein (Figures 1A and 1 B, lanes 5 and 6; data not shown). These data indicated that the POU domain alone was sufficient for dimerization. Deletion mutants that expressed either the POUHD or the PO& domain were separately incubated with the wild-type form of the other protein. These experiments revealed that the POUHD alone was sufficient to direct the formation of heterodimers and that the PO& domain was not required (Figures 1A and 1 B, lanes 7-10). The Role of Homeodomain Helical Structures in Dimerization The interaction between I-POU and Cfl-a was highly specific because I-POU failed to interact with Pit-l, Brn-1, Brn-2, Tst-1 , Ott-1 , or Ott-2 (data not shown). These observations led us to examine the amino acid sequences of the closely related Brn-2 and Cfl-a, which differ in the PO&, by three amino acids in the putative helix 1 (Figure 2A). The importance of these three amino acids in permittingadiscrimination between Brn-2and Cfl -adimerization with I-POU was assessed by mutating each of these residues in Brn-2, either singly, in pairs, or together, to the B. Cfl-a

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and Mutant

I-POU

and Cfl-a

Proteins

(A) Interaction of mutated I-POU proteins with Cfl-a. The stippled and darkened regions refer to the PO& domain and POUnD, respectively. The regions tested, their relative sizes, and their ability to form heterodimers with Cfl -a are indicated. The lower panel shows a typical experiment where Y34abeled WT I-POU or mutated I-POU proteins (N, POU, PO&, and PO&) are incubated with equimolar amounts of %labeled Cfl-a in the absence (-) or presence (+) of the chemical cross-linker, BMH (1 mM). The heterodimeric protein complexes are indicated with arrowheads. (8) Schematic representation of interaction of mutated Cfl-a proteins with I-POU-an analysis comparable with those in (A). Heterodimeric protein complexes are indicated with arrowheads. These experiments indicate that only the PO& is required for the heterodimeric interaction of I-POU and Cfl-a. Similar results were obtained in four independent experiments.

yll

of I-POU

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1 2 Figure

2. Specificity

of Protein-Protein

3 4

5

6

7 8

9 10 11 1213

14 15 16 17 18

Interaction

(A) Sequence comparison of the Cfl-a, Brn-2, I-POU, and Uric-86 POUHos. Of the POU-III proteins, Em-2 is most similar to Cfl-a, with only three amino acid differences within the entire POU HD, located specifically within the predicted first helix. I-POU does not form a dimeric complex with Em-2. Site-directed mutagenesis converted these three dissimilar amino acids, either singly, in pairs, or fully, to be identical to Cfl-a as shown in (B), and the resulting mutated proteins were assayed for their ability to interact with I-POU. The positions of indicated helices correspond to those predicted in I-POU. “WFC” refers to the three amino acids in helix 3 characteristic of POU domain proteins. (B) Interactions of mutant Brn-2 POU domains with I-POU. Brn-2 with the S-Q, L-H, C-Q, or combined mutations were evaluated for their ability to bind with I-POU and assayed by cross-linking analysis using 1 mM BMH (arrowhead indicating heterodimer, lane 18). Similar resultswere obtained in five independent experiments of similar design.

corresponding amino acid residues present in Cfl-a (see Figure 26). This analysis indicated that for I-POU to interact with the Brn-2 POU domain, it was necessary for all three amino acids in the Brn-2 POUHD helix 1 tocorrespond to those present in Cfl-a (Figure 28, lane 18). These data suggest that there is an absolute, specific sequence requirement within the first helix of the PO& for Cfl-a/ I-POU interaction. The role, if any, of sequence-specific information in helix 2 (H2) and helix 3 (H3) for Cfl-a/l-POU dimerization was examined by replacing these helices with the equivalent sequences from the ubiquitous octamer motif-binding protein, Ott-1 (Figure 3A). A BstEll site was introduced between helix 1 and helix 2 to permit substitution with helix 2 and helix 3 sequences of Ott-1. Introduc-

tion of this site altered three amino acids in the Cfl-a protein, but this three amino acid variant of Cfl -a protein was fully competent to dimerize with I-POU (Figure 38, lane 10). Therefore, the two amino acids (P and S) bordering helix 2 are apparently not directly involved in the Cfl-a/ I-POU interaction. However, the chimeric Cfl-a protein containing Ott-I helix 2 and helix 3 amino acid sequences was no longer capable of dimerizing with I-POU (Figure 38, lane 8), implicating the importance of specific sequences in helix 2 and/or helix 3. Because helix 3 of Ott-i and Cfl-a differed by only one amino acid residue (see Figure 3A), the possibility that helix 2, but not helix 3, provided a critical contact for the I-POUICfl-a heterodimer was considered. To examine di-

Cell 494

A

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1 Figure

3. Helical

Requirements

2

3

for I-POU

4

5

and Cfl-a

6 7

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11 1213

14 15 16

Interaction

(A) Introduction of prolines into either helix 1 (Hi-P), helix 2 (H2-P), or helix 3 (H3-P) of I-POU and Cfl-a. Chau-Fasman plots of these mutated proteins predict in all cases that the introduced prolines would destroy the a helix structure (not shown). Additionally, in the case of Cfl-a, the amino acids of helices 2 and 3 were replaced with those of helices 2 and 3 from Ott-1 protein (Ott-2/3), using a BstEll site created between helix 1 and helix 2 (see Experimental Procedures). (B) Protein-protein interactions were assessed using chemical cross-linking or BMH (1 mM) between wild-type Cfl -a/l-POU. Interaction was observed in the absence (-) or presence (+) of BMH as shown in lanes 1 and 2. In these experiments, quantitative interaction is shown by complete loss of both I-POU and Cfl-a bands and the appearance of cross-linked heterodimers (arrowhead). In all cases the cross-linked product was less intense than the two proteins that were not cross-linked. The effects of introduction of a proline into helix 2 or helix 3 of Cfl-a (lanes 3-6), of a BstEll site (lanes 9 and 10) and Ott-1 information into Cfl-a helices 2 and 3 (lanes 8 and 9), or of the introduction of proline residues into helix 1 (lanes 11 and 12), helix2(lanes ldand 14), or helix3(lanes IBand 16) areshown. Deletion ofthecfl-a helix3(removal ofthesequenceVRVWFCNRRQKEKR) failed to abolish heterodimer formation (data not shown). All results were confirmed in four separate experiments of identical design.

rectly this question and the role played by each of the putative POUHD helical regions in the I-POUICfl-a interaction, we introduced prolines into each of the helices of I-POU and Cfl -a at residues predicted by the Chau-Fasman algorithm (Chau and Fasman, 1974) to destroy the a helix, or deleted specific helices, and assayed the effects of each alteration on dimerization. The sites chosen in helices 1 and 2 correspond to residues that, when mutated in Pit-l, failed to alter DNA binding function, while the sites chosen in helix 3 abolished DNA binding function of Pit-l (Ingraham et al., 1990). Introduction of these proline residues into helix 1 and helix 2 of I-POU abolished its ability

to dimerize with Cfl-a (Figure 38, lanes 12 and 14). A similar result was obtained when a proline was introduced into helix 2 of Cfl-a (Figure 38, lane 4). Surprisingly, introduction of a proline residue into helix 3 of either I-POU or Cfl-a had no effect on dimerization (Figure 38, lanes 6 and 16), suggesting that the helical configuration of helix 3 was not required for dimerization. Furthermore, deletion of helix 3 of Cfl-a (from residue 47 through 60), required for its DNA binding activity, had no effect on Cfl-all-POU heterodimerformation. In contrast, deletion of helix 2 abolished this interaction (data not shown). Together, these data imply that the ability to direct the

yvil

of I-POU

formation of dimers is an intrinsic property of the first two helical structures (helix 1 and helix 2) and their specific primary sequence. In contrast, the “DNA recognition” helix, representing the most conserved sequences in the PO& region, appeared to exert little, if any, role in the dimerization event, implying that the crucial role of helix 3 is restricted to DNA binding events. t/-PO& An Alternatively Spliced Product of the I-POIJ Gene With the evidence that the POUHD alone was sufficient to provide the dimerization interface, the observation that I-POU was highly homologous (>95%) to Brn-3, a mammalian POU domain DNA-binding protein (He et al., 1989; Renge Gerrero and M. G. R., unpublished data) throughout the entire POU domain was enigmatic. What evolutionary pressure acted in the conservation of both the PO& domain and third helix of the POUHo in I-POU, a protein that does not bind specific DNA sequences? To begin to address this question, we examined the intron-exon genomic structure of I-POU in the POU domain coding region. Appropriate oligonucleotides corresponding to the C-terminal boundary of the PO& domain and the C-terminal boundary of the PO& were used to amplify DNA from Drosophila genomic DNA. This polymerase chain reaction (PCR) generated a ml.0 kb DNA fragment, while PCR with the same oligonucleotides using cDNA as template yielded the expected 200 bp fragment. The subsequent cloning and sequencing of the PCR-generated genomic DNA fragment indicated the presence of an intron in the middle of the basic cluster of residues N-terminal of the POUno (Figures 4A and 48). Examination of this structure revealed the presence of a consensus donor site, a polypyrimidine tract, a putative lariat branch point, and two potential S’consensus acceptor sites six nucleotides apart (Figure 4A). Potential splicing using these two acceptor sites would generate either I-POU or a second protein containing two additional basic residues (I-POU(+RK)), corresponding precisely to the additional two amino acids in this position in the brn-3 and uric-86gene products. This genomic structure was confirmed by analysis of cloned Drosophila genomic DNA. To test initially for the possible physiological utilization of this alternative splice site, we rescreened the Drosophila head cDNA library, from which I-POUcDNA was originally cloned. This screen yielded four positive clones that upon sequencing were shown to be fully identical over their entire 5’ and 3’ regions. However, within the PO&,, 2 of the 4 clones differed by the inclusion of the six additional nucleotides present immediately 5’ of the I-POU splice acceptor site (Figure 48; RHS). These data indicated that a splicing variant of /-POUthat utilized the more S’acceptor site was expressed. We have referred to this alternatively spliced variant as #POU. To examine more precisely the relative abundance of these spliced transcripts and to map their developmental expression, we performed RNAase protection analysis on RNA isolated from Drosophila at various stages. Utilizing a riboprobe from the tl-POUcDNA sequence encompassing the N-terminal basic region of the POUHD, we foundthatthe

largest protection fragment is consistent with full-length protection by the tl-POU mRNA sequence, while the two smaller fragments (190 and 40 nucleotides) correspond to scission of the probe at the mismatches generated by the absence of six nucleotides in the I-POU mRNA sequence. All three protection fragments were present throughout development, consistent with stochastic production of both I-POU and tl-POU mRNAs, without any apparent developmentally regulated splicing choice (Figure 4C). Thus, one genomic locus contained the coding sequences for two mRNAs encoding very similar but functionally distinct proteins: one, a non-DNA-binding factor capable of high affinity protein-protein interactions (I-POU); the other (tlPOU), a putative DNA-binding factor. tl-POU Does Not Interact with Cfl-a The unique structural distinction between I-POU and tlPOU, two additional basic residues in the N-terminal region of the POUHO, rendered tl-POU extraordinarily homologous to Brn-3. Therefore, the potential ability of tl-POU to dimerize with Cfl-a and to bind specific DNA sites was examined. Neither I-POUII-POU homeodimers nor I-POUI tl-POU heterodimers could be detected by cross-linking analysis under conditions where Cfl-all-POU heterodimers were readily detected (Figure 5A, lanes l-4; data not shown). Similar results were obtained using either the POU domain or the wild-type holoprotein (data not shown) of I-POU. When equimolar amounts of tl-POU and Cfl-a were incubated in the presence of chemical cross-linking agents, no heterodimer formation was observed (Figure 5A, lanes 5 and 6). The failure to form heterodimers was independently confirmed by immunoprecipitation analysis (data not shown), with I-POU providing a positive control, using specific anti-I-POU and anti-Cfl-a sera (Treaty et al., 1991). The unexpected inability of tl-POU to interact with Cfl-a clearly implicated a functional role for the basic cluster N-terminal of the POUHD in the dimerization of I-POU with Cfl-a. Interestingly, this basic cluster region exerts avital role in determining the DNA-binding potential of other POU domain proteins (Ingraham et al., 1990; Treaty et al., 1991). To investigate further the role of the basic amino acids of the N-terminus of the I-POU homeodomain, the effect of charge and spacing on dimerization and DNA binding activity was evaluated by mutagenesis (Figures 58, 5C, and 5D). While I-POU formed heterodimers with Cfl-a at a 1:l stoichiometry, I-POU with two additional basic residues&POU(+RK); i.e., tl-POU) now was incapableof such heterodimeric function (see Figure 5A, lane 6). When two alanine residues were introduced into I-POU at the identical position (LPOU(+2A)), this altered I-POU protein was capable of forming a dimer with Cfl -a, albeit with a remarkably reduced affinity (Figures 58 and 5C, lanes 3 and 4). These data indicated that it was both the altered spacing and the addition of two basic residues in tl-POU that precluded dimerization with Cfl-a. When all the N-terminal basic residues in I-POU were replaced with an identical number of alanine residues to maintain the spacing (Figure 58, I-POU(KKR+AAA)) or with acidic residues (Figure 58, (KKR+EEE)), no dimerization with Cfl-a was observed

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from the I-POU

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(A) The intron-exon boundaries are shown for the WOUgene in the N-terminal region of the POUnD. This approximate 800 bp intron (shown in part) contains a consensus donor site (underlined), two potential acceptor sites (underlined), a putative branch point (boxed), and an adenosine for lariat formation (indicated with an asterisk). This genomic structure can form two spliced products, as depicted: tl-POU and I-POU. (6) Sequence analysis of the genomic structure and cDNA products of the I-POU gene. The two cDNA clones (tl-POU and I-POD) were isolated independently from an adult Drosophila cDNA head library and displayed equal abundance (~2 clones1500,OOO pfu). The genomic sequence is shown on the left, and the sequences of the two cDNA clones are below. The two cDNA clones have identical DNA sequences throughout their 5’ and 3’ termini and diverge only by six nucleotides in the indicated region. (C) RNAase protections were performed at various embryonic instar, pupal, and adult stages (E = early: L = late). A 285 nucleotide antisense riboprobe encompassing the N-terminal region of the PO& was hybridized with 20 pg of total RNA isolated from Drosophila at all developmental stages. Hybridization with tl-POU should protect 236 nucleotides of probe. Hybridization with I-POU should protect 190 and 40 nucleotides of probe. The protected fragments for I-POU and tl-POU (labeled) indicated a stochastic production of both tl-POU and I-POU at all times in Drosophila development.

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123456

with Cfl-a

(A) tl-POU interactions with Cfl-a and I-POU. Chemical cross-linking experiments using BMH show heterodimers between wild-type I-POU and Cfl-a (lanes 1 and 2) but no dimer formation between I-POU (POU domain) and tl-POU (lanes 3 and 4) between I-POU holoprotein and tl-POU (data not shown), or between tl-POU and Cfl-a. Identical results were obtained in three independent experiments, (B) The effect of spacing and charge of dimerization. Two uncharged alanine residues were positioned in the center of the basic cluster (LPOU(+2A)), or all basic residues were removed from I-POU while maintaining the spacing with alanines (I-POU(KKR-AAA)) or with acidic residues (I-POU (KKR-EEE)). These mutated I-POU proteins were tested for their ability to dimerize with Cfl-a (see [C]) and to bind DNA (see [D]). A summary of the data in (C) and (D) is shown in the table. (C) Assays of these mutants for dimerization were tested by chemical cross-linking using BMH. The ability of I-POU(+2A) to dimerize with Cfl-a, with greatly reduced efficiency, was consistently observed. (D) DNA binding activity of mutant I-POU proteins. The ability of tl-POU(wt), I-POU(wt), I-POU(+PA), I-POU(KKR-AAA), and I-POU(KKR-EEE) to bind DNA was examined using gel shift assays and a total degenerate oligonucleotide (n = 16) as probe (Treaty et al., 1991). While tl-POU (i.e., I-POU(+RK)) was competent to bind a subset of the degenerate DNA-binding sites (lane 2) DNA binding by the three mutated I-POU proteins could not be detected (lanes 4-6) even with very long exposures. Similar results were obtained in three independent experiments.

Cell 498

(Figure 5C, lanes 5-8). These data revealed that a specific complement of charged residues present in the N-terminal region of the PO& was critical for dimerization, consistent with the possibility that dimerization involved both precise electrostatic and coiled-coil interactions between the two proteins. Thus, a specific sequence of the N-terminal basic region, in association with helix 1 and helix 2 of the POUHo, acts to coordinate protein heterodimerization. DNA Binding and Transcriptional Activity of tl-POU The potential ability of tl-POU to bind DNA was initially evaluated using a double-stranded oligonucleotide probe containing 18 contiguous fully degenerate positions, as previously described (Treaty et al., 1991), because the specific DNA-binding sites, if any, were unknown. This assay demonstrated that tl-POU could effectively bind DNA sites (Figure 5D, lane 2). Therefore, specific DNAbinding sites for several POU domain and classic homeodomain proteins were utilized in a gel shift assay (Figure 8A). These analyses indicated that tl-POU bound, with varying effectiveness, to all POU domain-binding sites tested; however, the Cfl-a site of the Ddc promoter appeared to be the lowest affinity site (Figure 6A). Thus, tl-POU was a DNA-binding factor, as predicted from its POU domain structure. The mutant forms of I-POU (see Figure 58) were utilized to examine what effect varying the charge and spacing present in the N-terminal region of the PO& of I-POU would have on DNA binding. I-POU(wt) failed to bind DNA in this assay, as expected, while tl-POU served as a positive control. Substitution of alanine residues for the two additional basic residues at the N-terminal portion of I-POU (+2A) did not result in ability to bind DNA (Figure 5D, lane 4). Similarly, substitution of the three basic residues on the N-terminus of the I-POUHo (with alanine or glutamic acid residues) failed to confer DNA binding activity (Figure 50, lanes 5 and 6). Thus, a specific sequence of the N-terminal basic region (in I-POU), in association with helix 1 and helix 2 of the POUHD, acts to coordinate protein heterodimerization, while aslightlyvariant N-terminal basic region (in tl-POU), in association with helix 3 of the PO&D, now coordinates DNA binding activity. To assay the transcriptional activation potential of tlPOU in vivo, we employed cotransfectional analysis using CV-1 cells (Figure 6B). While the natural target gene(s) of tl-POU during Drosophila neurogenesis remains unknown, cotransfection of tl-POU with various reporter genes under control of the binding sites previously examined in the gel shift assay (see Figure 6A) allowed evaluation of the potential of tl-POU to transactivate promoters containing these DNA-binding elements. tl-POU produced little, if any, activation of the Ddc reporter gene, consistent with its failure to bind significantly the Cfl-a response element and other neuronal-specific cis-active elements in this gene (M. N. T., unpublished data), while Cfl-a effectively transactivated the same promoter (Figure 6B). In contrast, the octamer-binding site (3 x OCT-L), to which tl-POU bound most avidly, reproducibly conferred a >l Ofold stimulation in response to tl-POU. tl-POU was almost as effective as Ott-2 on this element, while Cfl-a was

ineffective (Figure 6B). On promoters containing other elements, such as the Pit-l-binding site (Prl-1 P) or several homeodomain response elements, tl-POU exerted small or undetectable effects (Figure 6B). These data demonstrate that tl-POU can act as a transcription factor as a consequence of binding to specific cis-active elements and suggest that tl-POU can potentially activate a subset of genes distinct from those under the control of Cfl-a. Therefore, tl-POU and I-POU appear to exert complementaryfunctions, simultaneously activating and inhibiting two distinct sets of transcription units, respectively. Discussion Transcriptional Inhibition Resulting from Heterodimer Formation between POU Domain Proteins Dimerization interfaces of transcription factors distinct from, or overlapping, their core DNA-binding domains provide increased regulatory specificity in developmental and homeostatic control of gene expression. However, the initially characterized POU domain and classical homeodomain proteins were suggested to exist as monomers in solution. In the case of Pit-l and Ott-2, DNA site-dependent cooperative protein-protein interactions rely upon the PO& domain, as well as the POUHD, as has been observed on certain physiological and synthetic response elements (LeBowitz et al., 1989; lngraham et al., 1990; Voss et al., 1991). The observation that Pit-l is preferentially incorporated into Pit-l /Ott-1 heterodimers rather than homodimers (Voss et al., 1991) argues that heteromerit complexes on DNA might further modulate programs of gene activation. While there are clear examples of the control of transcription factor function through protein-protein interactions in solution (e.g., Baeuerle and Baltimore, 1988; Benezra et al., 1990; Ellis et al., 1990; Garrell and Modelell, 1990; Mudryj et al., 1991; Bandara and La Thangue, 1991; DefeoJones et al., 1991; Raychaudhuri et al., 1989), the specific interaction of I-POU with Cfl-a in solution provides an initial example of high affinity, DNA-independent interactions between members of the POU domain family of proteins (Treaty et al., 1991). The functional consequence of the I-POUICfl-a interaction is to prevent binding of Cfl-a to DNA recognition elements required for transactivating the Ddc gene. This provides a potential strategy for controlling developmental gene activation by a homeodomain-containing protein that does not bind DNA. The functions of I-POU within the context of the POU domain family of proteins iscomparablewith similar strategies used in other gene families. For example, Id, a member of the helix-loop-helix family of proteins, lacks basic amino acid residues and has no intrinsic DNA binding activity, but antagonizes the DNA binding activity of other members of the helix-loop-helix proteins through the formation of heterodimeric complexes in solution (Benezra et al., 1990). In Drosophila, putative interactions between members of the helix-loop-helix gene family appear to be responsible for neuroblast formation (Alonso and Cabrera, 1986; Caudy et al., 1988; Rushlow et al., 1989; Ellis et al., 1990; Garrell and Modolell, 1990). It has also been

grl

of I-POU

A

Binding

Site

OCT GH 1P ‘IPtlP”IPtlP”IPtlP’FiiF

Cfl

Binding

PO

Site

Ftz Eve ‘IPtlP”IPtlP”IP

Ubx

Free Probe -+

Free Probe +

B

Reporter

Ddc-L

Luciferase Activity (fold-induction)

+ Ex~~~~”

-1510 I

I-POUO

t1-POU Cfl -a 0 I-POU C-fla

3XOCT-L

“z2

3x1

+

P-L

L

0 I-POU [ t1-POU :~

Ubx-L Figure

6. DNA Binding

0

I-POU t1-POU

Ftz-L

and Transcriptional

Activity

of tl-POU

(A) The ability of I-POU (IP) and tl-POU (tlP) to bind DNA was examined using gel shift assays. Various DNA sites known to bind POU domain proteins (rGH, rat growth hormone Pit-l-binding site; IP, rat prolactin [Prl-1Pj Pit-l-binding site; Ott, octamer-binding site; Cf-1, Ddc promoter Cfl-a-binding site; and PO, Tst-IISCIP-binding site) on the PO promoter and also classic homeodomain DNA-binding sites (Ftz, fushi-tarazu-binding site; Eve, even-skipped-binding site; and Ubx, ultrabithorax-binding site) were utilized (see Experimental Procedures). tl-POU bound most sites, but with reproducibly different degrees of effectiveness, in the order Ott >> GH >> 1P > PO > Ftz - Eve - Ubx > Cfl. I-POU failed to show any DNA binding activity on any of these DNA sites. (6) Transcriptional activity of tl-POU. The transcriptional activity of tl-POU was examined in a series of cotransfection studies in CV-1 cells (mean + standard error of the mean). Luciferase reporter genes were under the control of the Ddc promoter (Ddc-L) or a minimal rat prolactin promoter -36 to 34 with three boxes of the Ott-binding site (3 x OCT-L). three boxes of the rat prolactin 1 P Pit-l-binding sites (3 x 1 P-L), one Ftz-binding site (Ftz-L), or one Ubx-binding site (Ubx-L). These promoters produced detectable luciferase activity when transfected into CV-1 ceils in the absence of expression vectors (between 50 and 500 light units per 100 ttg of whole cell protein). Cotransfection with I-POU expression vector (CMV I-POU) failed to activate any significant reporter gene expression. Cotransfection with tl-POU expression vector (CMV tl-POU) produced a 1O-fold stimulation of the octamer reporter gene expression (3 x OCT-L), but no significant stimulation of expression of the other reporter genes tested. Cotransfection of Cfl-a expression vector (CMV Cfl-a) produced the expected stimulation of the Ddc reporter gene expression (Ddc-L), but did not activate the 3 x OCT-L reporter gene. Coexpression of tl-POU and I-POU together at a 1 :l molar ratio did not affect the ability of tl-POU to activate the 3 x OCT-L reporter gene (data not shown). Similar results were obtained in three experiments.

Cell 500

suggested that the phosphoprotein IP-1, when dephosphorylated, could act as the dominant inhibitor of FosNun (AP-1) activity by specifically blocking DNA binding of AP-1 (Auwerx and Sassone-Corsi, 1991). Inhibition of nuclear transcription factor function can also operate at the level of competing DNA binding and/or partitioning of DNAbinding factors in the cytoplasm. For example, CREM, an antagonist of CREB-mediated transcription, acts by binding to the CRE site and down-regulating transcription (Foulker et al., 1991) which contrasts to cytoplasmic partitioning in control of NF-KB and certain ligand-dependent nuclear receptors (e.g., Groyer et al., 1987; Pratt et al., 1988; Baeuerle and Baltimore, 1988; Joab et al., 1984; Roth et al., 1989; Rushlow et al., 1989). Homeodomain-Mediated Heterodimeric Complex between Members of the POU Domain Gene Family The detailed analysis of the I-POUICfl-a dimerization interface, presented in this manuscript, has revealed several unexpected aspects of I-POUICfl-a heterodimer function. The region required for dimerization is entirely limited to the homeodomain and encompasses only the N-terminal basic amino acid cluster and helix 1 and helix 2. These datasuggestthatsimilarinhibitoryactionswill beobsewed in classical homeodomain proteins. A combination of specific charge, spacing, primary sequence, and a-helical structures in this region of the POUHo permits I-POU and Cfl-a to interact with a 1:i stoichiometry in solution. Disruption of helix 1 or helix 2 structure or alteration of precise amino acid sequences to those present in highly related POU domain protein sequences is sufficient to disrupt the dimerization event, implicating the important contribution of specific residues present in these two helices. Neither the PO& domain nor the POUHD DNA recognition helix 3 is required for the high affinity interaction between I-POU and Cfl-a. Within the POU domain family of proteins, helix 3 is the most highly conserved region (see Figure 7A) and is speculated to function in DNA, binding primarily through interactions with bases in the major groove (Kissinger et al., 1990; Otting et al., 1990). Nuclear magnetic resonance and X-ray crystallographic analyses of the classic homeodomain proteins show that helix 1 and helix 2 do not directly contact DNA and, therefore, in many cases, may serve other functions, including protein-protein interactions. Indeed, helix 1 and helix 2 regions in the POUHD show the greatest variability in residue content and would be predicted to provide a structural basis for the selective function in protein-protein discrimination between POU domain proteins (Figure 7A). The exquisite specificity of the I-POUICfl -a interaction is emphasized by the ability of I-POU to interact with Cfl-a, but not with Brn-2, a POU domain protein that differs by only three amino acids in the all within the first helix. This selective interaction POUHD, imposed by the homeodomain is consistent with analysis of the VPlG/aTIF interaction with Ott-1 (Gerster and Roeder, 1988; O’Hare and Goding, 1988; Stern et al., 1989; Kristie and Sharp, 1990) where the ability of VP1 61 aTlF to discriminate between the highly related Ott-1 and Ott-2 POU domain proteins was shown to involve the specific amino acid differences in helix 2 of the POUHD (Stern

et al., 1989). In this case, interaction with VP16, involving a third protein (e.g., Kristie and Sharp, 1990), does not effect Ott-1 DNA binding activity but exerts a role in transcriptional activation of specific genes in productive HSV infection. Within the classical homeodomain proteins, the residues of both helix 1 and helix 2, and the N-terminal basic region, are hypervariable (Scott et al., 1989) consistent with their potential selective roles in specific proteinprotein interactions. A remarkable feature of the I-POUI Cf 1-a interaction is that the ability of I-POU to form heterodimers is lost when the two basic amino acids uniquely absent in I-POU are restored in tl-POU, while the ability to bind DNA is reciprocally acquired. This reciprocal functional effect is instructive in light of the specific minor groove contacts by a basic amino acid in the N-terminus of the classic homeodomain proteins. While this region of the homeodomain is disordered in solution, it becomes ordered upon DNA binding (Kissinger et al., 1990; Otting et al., 1990). Dimerization of transcription factors is thought to involve specific coiled-coil interactions between specific helical structures as has been proposed in the case of bZIP, helix-loop-helix, and nuclear receptor gene families (e.g., Agre et al., 1989; Gentz et al., 1989; Glass et al., 1989; Kouzarides and Ziff, 1989; Landschulz et al., 1989; Murre et al., 1989; Schuermann et al., 1989; Turner and Tjian, 1989; Fawell et al., 1990). Our data indicate that both coiled-coil and electrostatic interactions are required for the Cfl-a/l-POU interaction. Examination of the charged residues in I-POU and Cfl -a suggests one potential model of the I-POUICfl-a interaction, in which helix 1 and helix 2 can be considered to interact in an antiparallel conformation, with the N-terminal basic residues of I-POU having electrostatic interactions with negatively charged residues located N-terminal to helix 3 of Cfl-a. Interestingly, the POU IV class uniquely lacks these acidic residues. Two of the critical specific residues in helix 1, as demonstrated by the Brn-2, Cfl-a study, would be oriented on the helical face away from DNA, if oriented according to the predictions of two classical homeodomain proteins (Kissinger et al., 1990; Otting et al., 1990). That the specific interactions of helix 1 and helix 2 may require precise register to occur is further suggested by the observation that tl-POU fails to form heterodimers with a mutant Cfl-a molecule in which two basic residues have been deleted from the N-terminus of the Cfl-a POUHD, comparable with the residues lacking in I-POU (M. N. T., unpublished data). Of course, such an antiparallel interaction would be in contrast to the more conventional view of parallel helical coiled-coil interactions, which has proven to be the mode of interaction between the leucine “zipper” of bZlP proteins (Landschulz et al., 1989; Gentz et al., 1989; O’Shea et al., 1989; Turner and Tjian, 1989). Direct physicochemical analyses will be required to test any model of I-POUICfl-a interactions. Cogeneration of Positive and Negative Transcriptional Regulators from the I-POU Genomic Locus The generation of two functionally distinct proteins, I-POU and tl-POU, as a consequence of alternative splicing

;$n

of I-POU

Class

III POU-Homeodomain I

I Cfl-a brn-2 brn-1 tst-1 Ceh-6

Class

GRKRKKRTSIEVSVKGALEQHFHKQPKPSAQEITSLADSLQLEKEWRVWFCNRRQKEKR -------------------S--L-C-----------------------------------------------------S--L-C----S----N------------------------------------G------S--L-C-----G-----G------------------------------------N--SR--F--QSNQ--N-----QV-ME--------------------III Hl H2

I-POU tI+O" brn-3

GEKK..RTSIAAPEKRSLEAYFAVQPRPSGEKIAAIAEKLDLKKNWRVWFCNQRQKQKR ----RK---------------------------------------------------------RK-----------------------S-----------------------------.D--RK----------E--QF-KQ---------DR---------------------

uric-86

I

II

Hl

B.

I-POU gene

1

H3

H2

EKKRKR

Tra?,

Mature Transcripts

H3

IV

Ryng

Cfl -a 111

Twin I-POU (DNA binding product)

tl-POU

target

(protein

I-POU binding

proaq

I-POU

: Cfl -a

gene(s)

Figure 7. Developmental Transcriptional Effects of I-POU (A) Homology between the POU domain proteins of class minimal variability of the N-terminus or helix 3 sequences. (B) Schematic representation of alternative splicing of the to inhibit Cfl-a-responsive genes or lo activate a distinct

Cfl-a and tl-POU Ill and class IV depicting

the marked

transcription

amino acid variability

WOU transcript and the divergent functions subset of specific target genes, respectively.

events from a specific genomic locus during Drosophila neurogenesis, has intriguing implications. Indeed, while the significance of alternative splicing of nuclear factors is generally unknown (e.g., Boggs et al., 1987; Bermingham and Scott, 1988; O’Connor et al., 1988; Busturia et al., 1990; lnoue et al., 1990; Leroy et al., 1991; Zelent et al., 1989), it is likely that generation of functionally distinct transcription factors from a specific gene may prove to be a commonly used mechanism in development. The

regulated

unit

in helix 1 and helix 2, with

of the I-POU and tl-POU

proteins,

serving

production of both I-POU and tl-POU during Drosophila neurogenesis suggests that the splicing machinery utilizes the two identified acceptor sites with equal

stochastic

efficiency

and without

preference.

tl-POU

is a transcription

factor, but appears to bind very poorly and function minimally on the Cfl -a response element in the Ddc gene promoter. While its physiological target genes remain to be elucidated, tl-POU constitutes a neuron-specific transcription factor that potentially regulates distinct sets of neu-

Cell 502

ronal genes. These actions of tl-POU are distinct from those of I-POU, which is a non-DNA-binding protein that antagonizes Cfl-a transcriptional activation by inhibiting it from binding DNA, as a consequence of the formation of highly specific and stable heterodimers in solution. Therefore, the I-POU gene generates both a specific inhibitor (I-POU) of one transcriptional program and, simultaneously, a specific activator (tl-POU) that is presumed to modulate a distinct pattern of gene expression (see Figure 76). The ability of the l-POUgene to generate simultaneously highly related proteins that may coordinate inactivation and activation of distinct programs of gene expression provides a potential mechanism for establishing sharp temporal and spatial boundaries in the appearance of specific cell phenotypes. The homeodomain-mediated interaction between I-POU and Cf 1-a is likely to be prototypic of regulatory heterodimeric interactions between other members of homeodomain-containing gene families. Experimental

Procedures

In Vitro Expression of Wild-Type and Mutant I-PO& tl-POU, and Cfl-a The pBSK II vector (Stratagene) containing the cDNA encoding for I-POU and Cfl-a, used in in vitro expression, has been described previously (Treaty et al., 1991). The cDNA encoding tl-POU was also cloned into this vector. These three constructs were used to generate all deletion mutants. Mutants of I-POU and Cfl-a, consisting of deletion of the C-terminus, were performed by introduction of a terminator codon and Sac1 restriction site at codons 201 and 257, respectively, by site-directed mutagenesis, using the procedure of Kunkel (1985). Subsequent restriction digestion with Sacl, gel purification of the fragment of interest, and religation of the vector produced the N-terminal coding region as shown in Figure 1. The deletions required to translate only the POU domain, PO& domain, and PO&, involved isolating the appropriate DNA sequence using specific primers and PCR. The 5’ primer always included an in-frame initiator methionine. The PCRgenerated DNA fragments were subcloned via Ndel-BamHI sites into modified T7 expression vector (pMET; Studier and Moffat, 1986) that contained a good Kozak sequence for translation initiation and a T7 terminator codon downstream of the inserted fragment (Kozak, 1989). For mutations containing deletions of the N-terminal region of Cfl-a (Figure 16, Cfl-a, C), introduction of a Hindlll site and downstream initiator methionine bysitedirected mutagenesis at codon 400allowed deletion of all N-terminal information. For generation of the Cfl-a/Ott-I construct, site-directed mutagenesis allowed the introduction of BstEll sites at the N-terminal boundary of helix 2 and the C-terminal boundary of helix 3. The excisable fragment was removed, and PCR was used to isolate the equivalent region from Ott-1 with in-frame BstEll sites. This Ott-1 fragment was subcloned into the Cfl-a plasmid, and sequence analysis confirmed the structure. The deletion of the Cfl-a helix 3 was at the V of VRVWFC through the end of the POUno. In most cases, plasmids were linearized (except pMET), and capped mRNA transcripts were prepared using a T7 or T3 RNA polymerase system, as previously described (Glass et al., 1989). These mRNAs were then used to program translation in a rabbit reticulocyle system in the presence or absence of [“Slmethionine. The radioactivity of the proteins translated in the presence of [“Slmethionine was determined by precipitation with trichloroacetic acid. All constructs were confirmed by sequence analysis, and two independent clones of each mutant were subjected to cross-linking analysis and SDS-PAGE. Plasmids used in transfectional analysis (I-POU, tl-POU, Cfl-a, Pit-l, and Brn-2) were placed in the polylinker of the pCMV1 expression vector and were preceded by the cytomegalovirus promoter region and followed by the human growth hormone termination and polyadenylation signal, as described (Elsholtz et al., 1990; lngraham et al., 1990; He et al., 1991; Treaty et al., 1991). The sequences of the oligonucleotides used in the mutational and PCR-generated constructs are as follows. Deletion

of the C-terminal region (using Sacl) of I-POU and Cfl-a, 5’-ACCGATCCXTGAGAGCTCGAGGCGTTT-3’ and 5’-ACTCCCACGTGAGAGCTCCTGGAGGCCJ’, respectively. For PCR isolation of the POU domain, the PO& domain, and POUHo regions of I-POU and Cfl-a for subcloning into pMET, the sequences are as follows. I-POU: N’-PO&, 5’-GATCCATATGGCTGGCCTGCATCCC-3’; N-PO&, 5’-GATCCATATGAAGCGGCGGGATCCG-3’; C-PO&, 5’-GATCGGATCCACTGGGCGCATCCGG-3’; C-POUHD, 5’-GATCGGATCCACGTTGATTGTGGCC-3’. Cfl-a: N-PO&, 51-GATCCATATGGGCGGCGGCGATCGG9’; N-PO&, 5”GATCCATATGTCCATTGACAAGATCGCC-3’; C-PO&, 5’-GATCGGATCCCTGAGCGGCGATCTT-3’; C-POUno, B”GATCGGATCCGTCGCCGCCGAGCGT-3’. For deletion of the N-terminal region of Cfl-a via Hindlll sites: 5’GATCAAGCTTGCCGCCATGGACGGCATGCCGCCG-3’. For the generation of the Cfl-a/Ott-1 chimeric gene using mutagenesis of Cfl-a (5’ of helix 2, 5’-CATAAGCAGCCGGTGACCTAAGCCCCAGGAGATA-3’; 3’ of helix 3, 5’-CAGAAGGAGAAGGTCACCACGCCGCCAAAT-37 and PCR of Ott-1 (5’ of helix 2, 5’-GCCGGTGACCACTATGATTGCTGATCAG-3’; 3’ of helix 3, B’CGGCGGTGACCGGGTTGAlTClTlllTC-37. All restriction sites are in bold type. Cross-Linking Cross-linking experiments were carried out as previously described (Treaty et al., 199’1). In brief, 2-5 ~1 of in vitro translated “S-labeled protein(s) was incubated in 20 pl of buffer (20 mM HEPES, 20% glycerol, 1 mM bismaleimiodohexane [BMH]) at room temperature for 20 min, and the reaction was quenched by the addition of 560 mM !3-mercaptoethanol and dye. Samples were heated to 95OC for 5 min and electrophoresed on a 12% or 15% discontinuous SDS-polyacrylamide gel. Molecular Cloning rl-POU was cloned from an adult Drosophila head lgtl0 cDNA library using [“PI/-POU cDNA as a probe. Recombinant plaques (1 x 106) transferred to nitrocellulose filters (Schleicher and Schuell) were hybridized (16 hr, 42%) in 50% formamide, 5x SSC, 5x Denhardt’s solution, 50 mM sodium phosphate (pH 7.0), and 100 &ml denatured salmon sperm DNAand washed to high stringency (0.1 x SSC, 65%). Four independent I-POUWPOU clones were isolated, and both sense and antisense strands were sequenced using oligonucleotide primers and T7 polymerase (Sequenase, USB). The DNA fragment generated from Drosophila genomic DNA by PCR using primers flanking the differential region of I-POUltl-POU was -806 bp larger than that obtained when PCR was performed on cDNA template, was cloned, sequenced, and used to screen a Drosophila lFix II genomic library. One 10 kb clone was isolated, and sequence analysis confirmed the presence of the intron. RNAase Protection Studies Specific primers and PCR were used to isolate a portion of the tl-POU cDNA. Thisfragment was subcloned in pBKSII, sequenced to confirm that no PCR-generated errors occurred, linearized with Smal, and T3 RNA polymerase was used to generate an antisense 285 nucleotide [y”P]UTP uniformly labeled probe, as previously described (He et al., 1989). Total RNA was isolated at various stages from Drosophila embryo, larva, pupae, and adult, and RNAase protection was performed as described (He et al., 1989). DNA Binding and Transfection Assays DNA binding studies were performed using standard gel retardation assays as described by Elsholtz et al. (1990). All binding assays were performed at room temperature for 20 min in a 12 ~1 volume of 4.0% ficoll, 25 mM HEPES (pH 7.8), 0.1 M KCI, 1 mM dithiothreitol, 0.01% NP-40. 2 pg of poly(dl-dC), 0.5 wg of bovine serum albumin, 0.25% nonfat milk, l-5 VI of in vitro translated protein, and 0.05-0.1 nM (0.5 nM for degenerate oligonucleotide) double-stranded oligonucleotide labeled with T4 polynucleotide kinase. Sequences of oligonucleotides used in these binding studies have been previously described (Ingraham et al., 1990; Treaty et al., 1991). One quarter of each reaction was loaded onto a 6% nondenaturing 0.5 x TBE-polyactylamide gel, electrophoresed at 500 V for 30 min, and autoradiographed for 6-l 4 hr at -80%. For transfection studies, Green monkey kidney cells (CV-1)

girl

of I-POU

were plated at a density of 0.5 x IO6 cells/60 mm plate in DMEM supplemented with 10% newborn calf serum. Twelve to twenty-four hours later, cells were transfected with 1 pg of expression vector(s) and 5 bg of reporter gene using the calcium phosphate coprecipitation method (Chen and Okayama, 1967). Cells were harvested 24 hr later and assayed for luciferase activity as described (deWet et al., 1967). The amount of plasmid used per plate was balanced in all experiments using pCMVneo. In all cases, two independent expression vectors were tested.

genesis and sex determination, has sequence similarities the achaete-scute complex. Cell 55, 1061-1067.

Acknowledgmenis

DefeoJones, D., Huang, P. S., Jones, R. E., Haskell, K. M., Vuocolo, G. A., Hanobik, M. G., Huber, Ii. E., and Oliff, A. (1991). Cloning of cDNAs for cellular proteins that bind to the retinoblastoma gene product. Nature 353, 251-254.

We thank Renee Gerrero, Catherine Godson, Chijen Lin, Jeff Voss, and Holly lngraham for helpful discussions, Charles Nelson for his critical assistance with transfectional analyses, and Rae Wu for her contributions to the genomic PCR analysis. For critical suggestions and comments we thank Drs. Robert Weinberg, Susan Taylor, and Charles Zuker. We also thank Charles Zuker for providing reagents. We gratefully acknowledge the Preuss Foundation for support. These studies were supported by grants from the American Cancer Society and the National Institutes of Health. M. N. T. is a research associate and M. G. R. is an Investigator with Howard Hughes Medical Institute. E. E. T. is a NARSAD Young Investigator and an HHMI Physician Research Fellow. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adveflisement” in accordance with 16 USC Section 1734 solely to indicate this fact. Received

September

13, 1991; revised

November

1, 1991

P. F., and McKnight, S. L. (1969). Cognate DNAretained after leucine zipper exchange between Science 246, 922-926.

Akam, M. (1967). The molecular Drosophila embryo. Development

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in the

Alonso, M. C.. and Cabrera, C. V. (1966). The achaete-scute gene complex of Drosophila melanogaster comprises four homologous genes. EMBO J. 7, 2585-2591. Auwerx, FoslJun 993.

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D. (1988). I-KB: a specific Science 242, 540-546.

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of

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